1. Field of the Invention
This invention pertains to a competitive chemiluminescent assay for the detection of the presence of cyclic nucleotide monophosphates. In general, the cyclic nucleotide phosphates appear only as monophosphates, due to chemical structure considerations. Among these, cyclic adenosine monophosphate (camp) is perhaps the most widely known, implicated as a primary or secondary messenger in a variety of cellular, intracellular and extracellular pathways. This invention takes advantage of the high sensitivity of chemiluminescent 1,2-dioxetane reagents to overcome sensitivity and dynamic range problems encountered in the prior art.
2. Background of the Prior Art
A wide variety of metabolic responses are keyed to release of intracellular cAMP. In many cases, these responses are mediated by cAMP-dependent protein kinase which, in the presence of elevated cAMP levels, triggers a wide variety of activating reactions. Among the best known metabolic responses keyed to cAMP are the conversion of glycogen to glucose in the liver, as well as a variety of activities keyed to the glycogen/glucose energy cycle. The principle hormone in this cycle which induces a rise in cAMP is epinephrine. There are, however, a wide variety of other hormones which will also trigger cAMP release, which in turn keys a metabolic response mediated by the kinase. These include adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), luteinizing hormone (LH). thyroid-stimulating hormone (TSH), parathyroid hormone, vasopressin and prostaglandin I. Accordingly, it is clear cAMP levels in specific tissues of mammals, including humans, may be key indicators of a variety of hormonal functions and interactions.
cAMP is only the best known of the cyclic nucleotides. In general, cyclic nucleotides appear as monophosphates. Guanosine monophosphate (cGMP), uridine monophosphate (cUMP) and cytidine monophosphate (cCMP) may all importantly bear on a wide variety of hormonal functions and intercellular interactions, that may be desirably measured. cAMP is the most studied of these xe2x80x9cmessengerxe2x80x9d cyclic nucleotides.
Assays, including competitive ELISA assays, for cAMP are known. A widely reported assay is available from Assay Design, and is a colorometric assay. Other immunoassay products are available from Amersham Med-Physics, Inc. (a radioimmunoassay) as well as IGEN and NEN. The Assay Design embodiment employs assay kits (also available under the mark Biomol(copyright) in a classic example of a competitive ELISA assay, in which the strength of the signal is inversely proportional to the concentration of the cyclic nucleotide present. The kit from Biomol is for measurement of light absorption. A fluorescent assay kit is available from Perseptive Biosystems.
Because of the very low values of cyclic nucleotides that may need to be detected in a variety of tissue samples, high sensitivity is required. Many of the commercially available assays for cAMP itself do not offer this sensitivity, and require acetylation of the cAMP (to promote better antibody binding) for greater sensitivity.
Accordingly, it remains a goal of those of skill in the art to find a competitive immunoassay which is highly sensitive, offers a broad dynamic range, and employs reagents which can be obtained through simplified procedures, including avoidance of acetylation.
The above objects, and others discussed in more detail below, are met by a chemiluminescent competitive ELISA assay which relies on the high chemiluminescent sensitivity of 1,2-dioxetanes. These dioxetanes, developed by the common assignee herein, Tropix, Inc., a division of P.E. Biosystems, are the subject of a wide variety of United States patents. The 1,2-dioxetanes that are useful in the claimed invention typically have a generic structure of Formula I: 
In Formula I, Y1, Y2 and X are variously electron-active moieties, in that they are either electron donating or electron withdrawing. Exemplary groups include halogens, particularly chlorine, alkoxies, particularly methoxy, amine, alkyl, etc. In the alternative, these groups are hydrogen. Any one or more of Y1, Y2 or X may be other than hydrogen, or they may all be hydrogen. Substituent R is an alkyl, aralkyl, cyclic alkyl, heteroalkyl comprising an O, N, P or S moiety, in general of less than 20 carbon atoms. Desirably, R is alkyl. R may be substituted with groups intended to enhance solubility as well as reactivity, which may include halogen substituents such as one or more fluorine atoms, carboxy (COO) substituents, sulfoxy substituents, etc. The same substituents to enhance solubility may also be present on Y1, Y2 or X. Ar is an aryl moiety, typically phenyl or naphthyl, most preferably phenyl. Z is a moiety which includes a bond which is cleaved by an enzyme, which, when cleaved, leaves either O or N attached to the aryl moiety. This anion destabilizes the dioxetane, leading to its decomposition. On decomposition, the dioxetane releases light. For the purposes of this invention, Z is a phosphate moiety, preferably disodium phosphate. Dioxetanes of this type are disclosed in U.S. Pat. Nos. 4,962,192; 4,931,569; 5,112,960; 5,145,772 and 5,654,154, as well as a variety of others. All of the foregoing patents are incorporated herein by reference. As disclosed, e.g., in U.S. Pat. No. 5,112,960, an enzyme-triggerable dioxetane such as 3-(4-methoxyspiro [1,2-dioxetane-3,2xe2x80x2-tricyclo[3.3.1.13,7] decan-4-phenylphosphate and its salts (AMPPD) is a highly effective reporter molecule of this type. Derivitisation of this xe2x80x9cunsubstitutedxe2x80x9d 1,2-dioxetane with substituents on the adamantyl ring, such as a chlorine atom (CSPD) can dramatically improve performance. Similarly, substituents on the phenyl ring besides the enzyme-cleavable substituent, particularly at the 3 or meta position, give further improved yields. These reporter molecules, which are chemiluminescent in nature, are referred to as enzyme-triggerable dioxetanes. The particular enzyme acting in the claimed invention is alkaline phosphotase, selecting therefore, Z as a phosphate moiety.
As set forth in U.S. Pat. No. 5,145,772 and 5,336,595, also incorporated herein by reference, the use of polymeric onium salts (ammonium, phosphonium and sulfonium) as enhancer molecules results in a heightened degree of light emission from the dioxetane on decomposition. This is due to the tendency of these macromolecular polymers to sequester the dioxetane anion, which is highly hydrophobic in non-aqueous xe2x80x9cmicro environmentsxe2x80x9d, which maximizes chemiluminescent emission. As further disclosed in U.S. Pat. No. 5,145,772, these dioxetanes may be combined with energy accepting fluorescent molecules, such as fluorescein, such that the energy released by the dioxetane on decomposition is transferred to the fluorescent receiver, the fluorescence being detected. The assay of this invention is particularly suited for chemiluminescent emission.
Neither the 1,2-dioxetanes, nor their preparation or use, per se, of enhancement agents, alone, or with secondary enhancement agents such as those taught in U.S. Pat. No. 5,547,836, constitute an aspect of the invention, per se.
In the assay of this invention, a microtiter well or similar xe2x80x9creaction chamberxe2x80x9d is coated with a capture antibody, which is exemplified by goat anti-rabbit IgG (available from a wide variety of sources, including American Qualex).
According to the invention, a conjugate of cAMP and alkaline phosphatase is prepared and added to the well with an antibody for cAMP (or other cyclic nucleotide) and the sample to be inspected (or standard). The reaction mixture is incubated, and washed. Thereafter, an alkaline phosphatase-triggerable 1,2-dioxetane, such as CSPD, is added, preferably in the presence of an enhancer, such as poly(vinylbenzyltributyl ammonium chloride) or other enhancement agents. The dioxetane is incubated, and the reaction chamber inspected, preferably with a luminometer, or some other type of light-sensitive device, for chemiluminescent signal. The stronger the signal, the lower the cyclic nucleotide concentration in the sample.